Method of amplifying an RNA target sequence using an RNA polymerase functioning preferably on RNA template

ABSTRACT

Disclosed is a process for transcribing RNA using a nucleotide reagent as the promoter. Such a reagent enables any type of RNA to be transcribed without sequence specification and without protein cofactors, by means of an RNA polymerase that is known to be DNA-dependent such as the RNA polymerase of the phage T7, or by means of new, mutated RNA polymerase with the ability to synthesize a transcription product of a polynucleotide matrix with a higher yield when the matrix is RNA than when said matrix is DNA. This type of RNA polymerase can be obtained by effecting mutations on a coding gene for a wild-type RNA polymerase, and then by selecting the mutated RNA polymerase with said ability. The invention can be applied notably to the detection, synthesis or quantification of RNA.

The subject of the invention is a method of transcription, which makesit possible to synthesize RNA strands complementary to an RNA template,as well as new RNA polymerases which make it possible to carry out thismethod.

The method of the invention leads to the amplification of RNA present insmall quantities in a biological sample, and thus allows the detectionand/or quantification of the RNA in the sample, or the sequencing of theproduct of amplification, in particular in the field of microbiology andvirology, and more generally in the field of medical diagnosis. Themethod of the invention may also be used in the synthesis of RNA probes.

It is known that in microbiology and in virology, the microorganismswhich it is sought to identify are often viable bacteria (thereforecontaining more RNA than DNA) or RNA viruses such as the HIV and HCVviruses. It is also known that in various pathologies, it is oftenadvantageous to monitor the variations in the expression of genes, andtherefore in the synthesis of messenger RNA.

It is therefore important to be able to have a simple and effectivemethod of amplifying an RNA target.

The PCR method, which makes it possible to cyclically amplify a DNAtarget, uses a single enzyme but requires the production of temperaturecycles, generally at three different temperatures. The PCR method may beadapted to the amplification of an RNA target by adding an additionalenzymatic activity of RNA-dependent DNA polymerase, which furthercomplicates this method.

The so-called NASBA/TMA method of amplification has the advantage ofbeing an isothermic method, but requires the use of three enzymaticactivities (RNA-dependent DNA polymerase, RNase H and DNA-dependent RNApolymerase) carried by two or three enzymes.

It is therefore desirable to be able to have a simple and automatablemethod of amplification of RNA, and in particular an isothermic methodusing only one enzyme.

To avoid the disadvantages, which have just been mentioned, of knownamplification techniques, it therefore appears to be necessary to use,for the amplification of RNA, an RNA-dependent RNA polymerase activity.

Unfortunately, the known natural RNA-dependent RNA polymerases (RNAdRNAp) are not suitable for such a use because they have specificrequirements as regards the RNA template, and their activity requiresthe presence of protein cofactors (also called auxiliary protein factorsor associated protein factors).

It has now been discovered that some known DNA-dependent RNA polymerasesare capable of transcribing a single-stranded RNA in the presence of adouble-stranded DNA promoter. Furthermore, some of these enzymes, whichare transformed by mutation, are capable of synthesizing atranscriptional product with a better yield when the template consistsof RNA than when the template consists of DNA.

In the present application, the term “transcription” designates thesynthesis of several strands of RNA in the presence of a polynucleotidetemplate and of ribonucleoside triphosphates, in an appropriate reactionmedium and under conditions allowing the catalytic activity of an RNApolymerase to be exerted. The transcription occurs by synthesis of acomplementary or antiparallel copy of the template. The strand of thetemplate which is copied is called the transcribed strand or thetemplate strand. The synthesis of the RNA progresses in the 5′-3′direction.

It is known that some RNA polymerases function under the control of apromoter. A promoter is a double-stranded nucleotide sequence recognizedfor the RNA polymerase and necessary for the initiation oftranscription.

It should be recalled that when the template strand is linked to thepromoter, the first nucleotide transcribed on the template strand,linked by its 3′ end to the 5′ end of one of the strands of thepromoter, is designated by +1. The strand of the promoter which islinked to the template strand is called the antisense strand. The otherstrand of the promoter, which is complementary to the antisense strand,and hybridized to it, is called sense strand. The successive nucleotideswhich are situated on the side of the promoter, with respect tonucleotide +1, are, starting from +1, numbered −1, −2, −3, and the like.

The position −1 therefore corresponds to the 5′ end of the antisensestrand of the promoter, and to the 3′ end of the sense strand. However,some authors include the nucleotide sequence corresponding to the regionwhere the transcription starts (in particular the sequence from +1 to+6, for which a consensus sequence can generally be defined) in thedefinition of the sequence of the promoter.

On the template strand, the positions of the successive nucleotidescopied, starting from +1, and therefore in the 3′-5′ direction, arenoted +2, +3, and the like.

In the text which follows, the terms sense strand and antisense strandare generally used for the promoter itself (positions numberednegatively), and the term non-template strand is used for any strandlinked to the 3′ end of the sense strand, and the term template strandfor any strand linked to the 5′ end of the antisense strand or for anystrand hybridized to the non-template strand. In a given polynucleotidestrand, “upstream region” refers to a region situated on the side of the5′ end, and “downstream region” a region situated on the side of the 3′end. However, in the domain of transcription under the control of apromoter, and without taking into consideration a particular strand,“upstream” region traditionally refers to the region which, relative toposition +1, is on the side of the promoter (positions indicated bynegative numbers), and “downstream” region the region situated on theside of the template copied (positions indicated by positive numbers),such that the downstream direction then corresponds to the 3′-5′direction on the template strand, and to the 5′-3′ direction on thenewly-synthesized RNA strand.

The template strand is not necessarily linked to the 5′ end of theantisense strand of the promoter. However, it should, in this case, behybridized to a complementary and antiparallel strand (non-templatestrand) which is itself linked by its 5′ end to the 3′ and of the sensestrand of the promoter; see ZHOU W. and DOETSCH P. W., Biochemistry 33,14926-14934 (1994) and ZHOU W. et al., Cell 82, 577-585 (1995). In sucha case, the transcription may start in any position, which may rangefrom +1 to +24, corresponding to the 3′ end of the template strand or ofthe part of the template strand hybridized with the non-template strand.

Compared with bacterial, eukaryotic or mitochondrial RNA polymerases,the phage RNA polymerases are very simple enzymes. The best known amongthem are the RNA polymerases of the T7, T3 and SP6 bacteriophages. Thebacteriophage RNA polymerase has been cloned; see in particular U.S.Pat. No. 4,952,496. These enzymes are highly homologous to one anotherand consist of a single subunit. The natural promoters specific for theRNA polymerases of the T7, T3 and SP6 phages are well known. Thesequencing of the whole genome of the bacteriophage T7 (Dunn et al., J.Mol. Biol. 166, 477-535 (1983)) has made it possible to define theexistence of 17 promoters on the DNA of this phage. Comparison of these17 sequences shows that 23 contiguous nucleotides situated betweenpositions −17 and +6 relative to the site of initiation (position +1) oftranscription, are highly conserved. These nucleotides are evenidentical in five so-called class III promoters, which are the mostefficient in particular in vitro. Likewise, many promoter sequencesspecific for the T3 RNA polymerase also exhibit a very high homology, inparticular between positions −17 and +6. Moreover, several differentsequences of promoter for phage SP6 RNA polymerase have been identifiedand also exhibit a high homology; see Brown J. E., et al., Nucleic AcidsRes. 14, 3521-3526 (1986).

It is therefore possible to consider that the various phage RNApolymerases mentioned above form part of a family of RNA polymeraseswhich recognize promoters having a consensus sequence from position −17to position +6, and in particular from position −17 to position −1.

The method of the invention makes it possible to transcribe any RNAsequence because the RNA polymerases, capable of transcribing RNA underthe control of a promoter, which are described in the presentapplication, may transcribe RNA without a high sequence specificity. Itis known, nevertheless, that some sequences for initiation oftranscription, in particular from position +1 to position +6, are morefavorable than others for obtaining transcripts of the expected lengthwith a given phage RNA polymerase, in the case of the transcription ofDNA; see for example Milligan J. F. et al., Nucleic Acids Research, 15,8783-8798 (1987). The RNA polymerases capable of transcribing RNA whichare described in the present application may also function with variableyields according to the sequence of the region for initiation oftranscription. The sequences which are most suitable for a given RNApolymerase may be determined, where appropriate, by simple routineexperiments similar to those described by Milligan et al. in the articlewhich has just been mentioned. In addition, as will be seen below, themethod of transcription of the invention makes it possible, whereappropriate, either to start the transcription in a favorable region ofthe RNA to be transcribed, or to provide a reagent-promoter whichalready contains a region for initiation of transcription having asequence favorable for a given RNA polymerase.

The subject of the present invention is therefore a method of amplifyingany RNA target sequence, by transcription under the control of apromoter, in an RNA sample comprising said target sequence, in whichsaid sample is brought into contact:

with a reagent capable of hybridizing with said RNA comprising saidtarget sequence,

and with an enzymatic system comprising an RNA-dependent RNA polymeraseactivity, under conditions allowing the hybridization of said reagentwith said RNA comprising said target sequence and under conditionsallowing the functioning of said RNA-dependent RNA polymerase activity;in which said reagent contains:

(i) a first nucleotide strand comprising: a) a first nucleotide segmentcapable of playing the role of sense strand of a promoter for said RNApolymerase activity and b), downstream of said first segment, a secondnucleotide segment comprising a sequence capable of hybridizing with aregion of said RNA, and

(ii) in the hybridized state on the first strand, a second nucleotidestrand comprising a third nucleotide segment capable of hybridizing withsaid first segment so as to form with it a functional double-strandedpromoter;

and in which said RNA polymerase activity is capable of transcribing anRNA template, in the presence of said reagent hybridized with saidtemplate, in the absence of associated protein factor and in the absenceof a ligase activity.

The general conditions allowing the hybridization of nucleotide strandsare known, and specific conditions may be easily determined, by routineexperiments, for strands of a given sequence. The conditions allowingthe functioning of the RNA polymerase activity, in the presence ofribonucleoside triphosphates, may also be easily determined byexperiments, optionally with the aid of the information provided in theexperimental section below.

The 3′ end of the first segment corresponds to position −1 in thetranscriptional system used. The first segment contains a sufficientnumber of nucleotides to be able, in the hybridized state, to play therole of a promoter for an RNA polymerase. According to a specificembodiment, the first segment contains at least 9 nucleotides.

In patent FR 2,714,062, it has been shown that short sequences of 6 to 9consecutive nucleotides chosen from the −12 to −4 region of the sensestrand of a promoter for a phage RNA polymerase are capable of playingthe role of functional promoters in the transcription of a DNA targetsequence.

The reagent used in the method of the invention may also exhibit atleast one of the following characteristics:

said third segment is flanked, at its upstream end, by a fourthnucleotide segment which is shorter than said second segment of thefirst strand;

said fourth segment is capable of hybridizing with a portion oppositesaid second segment.

said first and third segments consist of DNA;

said third and fourth segments consist of DNA or RNA.

The third segment may have the same length as the first segment. It mayalso be shorter or longer, but its 5′ end must correspond to position −1(that is to say the position immediately preceding the position forinitiation of transcription in the case where the template strand islinked to the promoter), when it is hybridized with the first segment.

When the second strand of the reagent does not contain the fourthsegment, the reagent may be used in particular to transcribe an RNAwhose 3′ end region, or a region close to the 3′ end, has a knownsequence, and in this case the second nucleotide segment of the firststrand is constructed so that said RNA, in the vicinity of its 3′ end,is capable of hybridization with at least part of the sequence of saidsecond nucleotide segment. The 3′ end of the part of the RNA to betranscribed which is hybridized to the second segment may be contiguousto the 5′ end of the third segment, or it may be distant therefrom by anumber x of nucleotides (counted on the second segment), x representingzero or an integer from 1 to 24. Of course, the length of the secondsegment (in number of nucleotides) is greater than x, in order to beable to ensure the binding of the RNA template to be transcribed, byhybridization with a downstream region of the second segment.

The fourth segment, containing for example from 1 to 18 nucleotides, andin particular from 1 to 12 nucleotides, preferably has a sequence chosenso as to favor the initiation of transcription for a given RNApolymerase (see in particular the experimental section below). Thefourth segment may be produced in particular in DNA. Its sequence may becomplementary to the upstream region of the second segment facing it andto which it is then hybridized. In this case, the choice of the sequenceof the 5′ region of the second segment is dictated by the choice of thesequence of the fourth segment. It is not necessary for the fourthsegment to be linked to the third segment since in any case its correctpositioning in order to favor the initiation of transcription may beensured by its hybridization to the second segment. However, in aspecific embodiment, the fourth segment is linked to the third segment.

As above, the 3′ end of the target RNA part which is hybridized with thesecond segment may be distant from the 5′ end of the fourth segment by anumber of nucleotides equal to x, as defined above.

For obvious reasons, the second segment contains a number of nucleotidesat least equal to the sum of the number of nucleotides of the fourthsegment, if it is present, and of the number of nucleotides of saidsequence of the second segment which is capable of hybridizing with saidregion of the RNA to be transcribed.

The method of transcription of the invention may be carried out with avirus or phage wild-type RNA polymerase, and in particular with an RNApolymerase chosen from the family of RNA polymerases, mentioned above,which includes the T7 RNA polymerase, T3 RNA polymerase and SP6 RNApolymerase.

It has indeed been discovered that these RNA polymerases, known to beDNA-dependent, were also capable of transcribing an RNA template,optionally choosing (for example by virtue of the fourth segmentdescribed above) a sequence favoring the initiation of transcription.

The discovery of this RNA-dependent RNA polymerase activity makes itpossible to have for the first time RNA polymerases capable oftranscribing RNA under the control of a promoter, starting from position+1, and in the absence of associated protein factor.

It is also possible to carry out the method of the invention withmutated RNA polymerases which will be described in greater detail below.The importance of these mutated RNA polymerases is that some of them arecapable of carrying out the transcription with a better yield when thetemplate consists of RNA than when the template consists of a comparableDNA (that is to say containing deoxyribonucleotides A, C, G in place ofthe ribonucleotides A, C, G, respectively, and containing thedeoxyribonucleotide T in place of the ribonucleotide U).

The invention also relates to the use of an RNA polymerase capable oftranscribing an RNA template, under the control of a promoter, in theabsence of, auxiliary protein factor, in a method of transcription of atemplate strand comprising an RNA target sequence in which said RNApolymerase is chosen from the T7 RNA, the SP6 RNA polymerase and themutated RNA polymerases as defined above. The template strand mayconsist of RNA, or may consist of DNA in the region for initiation oftranscription, and then of RNA next.

The invention also relates to the use of an RNA polymerase capable oftranscribing an RNA template, under the control of a promoter, in theabsence of auxiliary protein factor, in a method of transcription of atemplate strand comprising an RNA target sequence, in which saidtemplate strand consists of RNA at least between position +5 and the 5′end of the target. The template strand therefore consists of RNAstarting from one of the positions +1 to +5, and may therefore consistof DNA from position +1 to position +2, +3 or +4. The article by S.Leary et al. mentioned below describes the transcription of a templateconsisting of DNA for positions +1 to +6 and then of RNA for positions 7and the next ones, with T3 RNA polymerase.

The invention also relates to the RNA-dependent RNA polymerases(RNAdRNAp) obtained by modification of DNA-dependent RNA polymerases andwhich are capable of synthesizing RNA strands complementary to an RNAtemplate. They may be used, for example, in the sequencing of RNA, thesynthesis of RNA probes, and amplification techniques allowing inparticular the detection and quantification of RNA.

The known natural RNAdRNAp are not suitable for use as polymerases inseveral applications because they have acquired a strong discriminatorycapacity with respect to their specific RNA template. Furthermore, theseenzymes have not been well characterized. For the majority, they formsupramolecular complexes composed of both viral and cellular factors;these complexes, which are generally associated with the membranes, aredifficult to purify and are unstable during isolation (B. N. Fields, D.M. Knipe, Virology, Vols 1 and 2, Raven Press, New York, (1990); G. P.Pfeifer, R. Drouin, G. P. Holmquist, Mutat. Res. Fundam. Mol. Mech.Mutagen. 288, 39 (1993)).

Few RNAdRNAp have been cloned, sequenced and expressed. The enzyme Qβreplicase is the best characterized. This enzyme is composed of 4subunits, of which 3 are host factors (M. Kajitarii, A. Ishihama,Nucleic Acids Res. 19, 1063 (1991)). The Qβ enzyme has been isolated; itshows good processability and is capable of carrying out cyclicreactions (P. M. Lizardi, C. E. Guerra, H. Lomeli, I. Tussie-Luna, F. R.Kramer, Bio/Technology 6, 1197 (1988)). However, this enzyme remainsvery limited in its applications because it recognizes as template onlya restricted class of highly structured RNA molecules (V. D. Axelrod, E.Brown, C. Priano, D. R. Mills, Virology 184, 595 (1991)).

Another RNAdRNAp has been partially characterized. It is an enzyme fromthe Saccharomyces cerevisiae L-A virus. This polymerase, which has beencloned, requires assembling of the viral particle; it binds first of allto the plus RNA strand, and then induces the assembly of the proteins ofthe particle (T. Fujimura, R. Esteban, L. M. Esteban, R. B. Wickner,Cell 62, 819 (1990)). At least three factors are known to combine withthe viral particles. These factors are necessary for the replication ofRNA, for transcription and for the coherent maintenance of the particle(T. Fujimura, R. B. Wickner, Molec. Cell. Biol. 7, 420, (1987)). Studiesin vitro have shown that an intact viral particle is necessary for thesynthesis of the minus strand (replication) (T. Fujimura, R. B. Wickner,Cell 55, 663 (1988)) and for the synthesis of the plus strand(transcription) (T. Fujimura, R. B. Wickner, J. Biol. Chem. 264, 10872(1989)). Thus, the complexity of this system does not make it easilyadaptable to an in vitro transcription system. Furthermore, just likethe Qβ system, this system is very discriminatory, accepting only M andL-A viral RNAs as template (T. Fujimura, R. B. Wickner, Cell 55, 663(1988)).

An RNAdRNAp for which a broad template accepting capacity has been shownis the enzyme of the poliomyelitis virus (J. Plotch, O. Palant, Y.Gluzman, J. Virol. 63, 216 (1989)). However, several problems exist withthis enzymatic system: the priming is dependent either on anunidentified host factor or on the addition of a poly(U)oligonucleotide. However, given that priming with a poly(U)oligonucleotide is not selective with respect to the template, manyproducts of different sizes are synthesized, in particular productshaving twice the length of the template. Furthermore, the sequentialsynthesis of the plus and minus strands has not been demonstrated (S. J.Plotch, O. Palant, Y. Gluzman, J. Virol. 63, 216 (1989), T. D. Hey, O.C. Richards, E. Ehrenfeld, J. Virol. 61, 802 (1987), J. M. Lubinski, L.J. Ransone, A. Dasgupta, J. Virol 61, 2997 (1987)).

Among the DNAdRNAp polymerases, the enzymes of the T3 and T7bacteriophages are capable of using RNA as template under particularconditions. For example, the T3 DNAdRNAp may transcribe asingle-stranded RNA template (i.e. the messenger RNA for the gene forresistance to neomycin) if it is ligated to the antisense strand of theT3 promoter including the sequence for initiation from +1 to +6 (S.Leary, H. J. Baum, Z. G. Loewy, Gene 106, 93 (1991)). It is also knownthat the T7 RNA polymerase can transcribe, from one end to the other, anRNA template in the absence of the promoter sequence (M. Chamberlin, J.Ring, J. Biol. Chem. 248, 2235 (1973)). Furthermore, it has been shownthat the T7 RNA polymerase can efficiently transcribe two small specificRNA templates, the “X” and “Y” RNAs, producing both plus and minus RNAcopies. This replication, which is obtained in the absence of aconsensus promoter sequence, appears to be dependent on the presence ofa specific secondary structure (M. M. Konarska, P. A. Sharp, Cell 57,423 (1989), M. M. Konarska, P. A. Sharp, Cell 63, 609 (1990)). On theother hand, the “X” and “Y” RNAs are not replicated by the T3 RNApolymerase, and it is not known if this enzyme is not capable ofreplicating highly structured RNAs, or if the sequence specificity ofthis enzyme prevents its recognition of the “X” and “Y” RNAs. In theabsence of a promoter, it has also been shown that the T7 DNAdRNAp wascapable of carrying out the extension of two overlapping RNA strands inantisense (C. Cazenave and O. C. Ulhlenbeck, Proc. Natl. Acad. Sci. USA91, 6972 (1994)). Likewise, it has been shown that the wild-type T7DNAdRNAp is capable of carrying out the extension of an RNA primer on asingle-stranded DNA template, in the absence of a promoter (S. S. Daubeand P. H. von Hippel, Biochemistry 33, 340 (1994)).

The best characterized bacteriophage enzyme is the T7 RNA polymerase, amonomeric enzyme of 98 kDa (B. A. Moffatt, J. J. Dunn, F. W. Studier, J.Mol. Biol. 173, 265 (1984)). This monomeric polymerase has all theessential properties of an RNA polymerase, that is to say recognition ofa promoter, initiation of transcription, extension and termination (M.Chamberlin, T. Ryan, The Enzymes XV, 87 (1982)). Furthermore, thecatalytic activity requires few elements, namely a template,ribonucleoside triphosphates and the divalent Mg²⁺ ion, and it does notrequire any auxiliary protein factor for the initiation or terminationof transcription, unlike the other RNA polymerases (M. Chamberlin, T.Ryan, The Enzymes XV, 87 (1982)).

Mutagenesis of the T7 RNA polymerase gene has made it possible toidentify and to define regions or residues involved in the polymerasefunction. A mutagenesis strategy has consisted in exchanging elementsbetween the T7 RNA polymerase and its close relative the T3 RNApolymerase whose amino acid sequence is 82% identical (K. E. Joho, L. B.Gross, N. J. McGraw, C. Raskin, W. T. McAllister, J. Mol. Biol. 215, 31(1990)). This strategy has led to the identification of polymeraseelements involved in the recognition of the promoter. It has been shown,for example, that the substitution of a single amino acid in the T3 (orT7) enzyme allows the mutated enzyme to specifically recognize theheterologous T7 (or T3) promoter (C. A. Raskin, G. Diaz, K. Joho, W. T.McAllister, J. Mol. Biol. 228, 506 (1992)). In the same manner,reciprocal substitutions in the respective promoter sequences confer onthe mutated promoter the capacity to be recognized by the heterologousenzyme (C. A. Raskin, G. Diaz, K. Joho, W. T. McAllister, J. Mol. Biol.228, 506, (1992)).

T7 RNA polymerase has been crystallized and its structure determined ata resolution of 3.3 Å (R. Sousa, Y. J. Chung, J. P. Rose, B.-C. Wang,Nature 364, 593 (1993)). From this structural study, sequence alignments(K. E. Joho, L. B. Gross, N. J. McGraw, C. Raskin, W. T. McAllister, J.Mol. Biol. 215, 31 (1990); S. Mungal, B. M. Steinberg, L. B. Taichman,J. Virol. 66, 3220 (1992), W. T. McAllister, C. A. Raskin, Molec.Microbiol. 10, 1 (1993)) and mutagenic studies (D. Patra, E. M. Lafer,R. Sousa, J. Mol. Biol. 224, 307 (1992); L. Gross, W-J. Chen, W. T.McAllister, J. Mol. Biol. 228, 1 (1992)), it has been possible tocorrelate the functional elements of the T7 enzyme with the structuralelement. T7 RNA polymerase may be divided into two functional domains: apromoter recognition domain and a catalytic domain (R. Sousa, Y. J.Chung, J. P. Rose, B. -C. Wang, Nature 364, 593 (1993), W. T.McAllister, Cell. Molec. Biol. 39, 385 (1993)).

The T7 RNA polymerase asparagine 748 has been shown to interact withnucleotides −10 and −11 in the promoter sequence, an interaction shownto be responsible for the promoter specificity (C. A. Raskin, G. Diaz,K. Joho, W. T. McAllister, J. Mol. Biol. 228, 506 (1992). Thepossibility that a sigma-type interaction between the T7 polymerase andits promoter can exist in the bacteriophage system has been mentioned.Indeed, a sigma-type sequence, corresponding to the 2.4 region of sigma,i.e. the region of sigma interacting with the “Pribnow box” (TATAATGsequence recognized by the E.coli sigma 70 transcription factor) (C.Waldburger, T. Gardella, R. Wong, M. M. Susskind, J. Mol. Biol. 215, 267(1990); D. A. Siegele, J. C. Hu, W. A. Walter, C. A. Gross, J. Mol.Biol. 206, 591 (1989)), exists in the N-terminal region of the T7 RNApolymerase between amino acids 137 and 157 (L. Gross, W -J. Chen, W. T.McAllister, J. Mol. Biol. 228, 1 (1992)). Moreover, although it has notbeen possible to attribute any function to it, the 230 to 250 regionexhibits sequence homologies with the E.coli λ repressor (McGraw, N. J.,Bailey, J. N., Cleaves, G. R., Dembinski, D. R., Gocke, C. R., Joliffe,L. K., MacWright, R. S. and McAllister, W. T. Nucleic Acids Res. 13,6753 (1985)).

The catalytic domain consists of a pocket resulting from the bringinginto close proximity of several regions dispersed over the primarystructure (R. Sousa, Y. J. Chung, J. P. Rose, B.-C. Wang, Nature 364,593 (1993), W. T. McAllister, C. A. Raskin, Molec. Microbiol. 10, 1(1993); D. Moras, Nature 364, 572 (1993)). This pocket contains inparticular several conserved motifs among which the A and C motifs arethe best conserved in the polymerases (Poch, O., Sauvaget, I., Delarue,M. and Tordo, N. EMBO J. 8, 3867 (1989); Delarue, M., Poch, O., Tordo,N. and Moras, D. Protein Engineering 3, 461 (1990); W. T. McAllister, C.A. Raskin, Molec. Microbiol. 10, 1 (1993)). A third motif, the B motif,is conserved in DNA-dependent RNA and DNA polymerases whereas a B′ motifwhich is different (both for the sequence and for the apparentstructure) exists in the RNA-dependent RNA and DNA polymerases (Poch,O., Sauvaget, I., Delarue, M. and Tordo, N. EMBO J. 8, 3867 (1989);Delarue, M., Poch, O., Tordo, N. and Moras, D. Protein Engineering, 3,461 (1990); L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice, T. A.Steitz, Science 256, 1783 (1992); W. T. McAllister, C. A. Raskin, Molec.Microbiol. 10, 1 (1993)).

One of the aspects of the present invention is based on the discoverythat certain mutated DNA-dependent RNA polymerases are capable oftranscribing a single-stranded or double-stranded RNA in the presence ofa double-stranded DNA promoter. Furthermore, these mutant enzymes arenot very capable or are incapable of transcribing single-stranded ordouble-stranded DNA in the presence of a double-stranded DNA promoter.They are therefore preferably or strictly RNA-dependent. Their use isparticularly advantageous in cases where it is desired to selectivelytranscribe the RNA, in particular when the starting biological samplecontains or risks containing DNA having a sequence identical or similarto that of the RNA to be amplified.

The subject of the invention is therefore an RNA polymerase capable oftranscribing a polynucleotide segment of interest of any sequencecontained in a polynucleotide template, by synthesizing, in the presenceof said template, and under the control of a promoter, a product oftranscription containing an RNA sequence complementary to the sequenceof said polynucleotide segment of interest, characterized in that it iscapable of synthesizing said product of transcription with a betteryield when said sequence of interest contained in the template consistsof RNA than when said sequence of interest contained in the templateconsists of DNA.

The invention relates in particular to an RNA polymerase defined asabove such that the ratio of the yield of product of transcription of aDNA template to the yield of product of transcription of an RNAtemplate, expressed in %, is less than 95%, especially less than 85% andin particular less than 70%.

The subject of the invention is in particular an RNA polymerase asdefined above, characterized in that the ratio of the yield of productof transcription of the RNA template to the yield of product oftranscription of the DNA template is at least equal to 2 and inparticular at least equal to 10.

The “yield” of transcription is the molar ratio of the quantity ofproduct of transcription to the quantity of polynucleotide templatepresent at the origin. This yield may be easily determinedexperimentally, by introducing into the reaction medium a determinedquantity of the polynucleotide template. For comparison of the yieldsobtained with a DNA template and an RNA template, conditions other thanthose of the nature of the template must obviously be comparable.

The RNA polymerase of the invention is capable of transcribing apolyribonucleotide template of any sequence, and it differs fromQβ-replicase in this respect. It preferentially or exclusivelytranscribes an RNA template, and it differs from known phageDNA-dependent RNA polymerases in this respect.

The RNA polymerases of the invention, unlike the known natural RNAdRNAPpolymerases, are in particular RNA polymerases capable of functioningwithout associated protein cofactor(s). They may however be provided inthe form of multimers, and in particular of dimers.

The mutated RNA polymerases of the invention are therefore generallyobtained from RNA polymerases which are themselves capable offunctioning without protein cofactors.

The RNA polymerases of the invention may be in particular RNApolymerases which are derived by mutation from a virus or phageDNA-dependent RNA polymerase, and in particular from a DNA polymerase ofan E. coli phage. Among the E. coli phages, there may be mentioned inparticular T3, T7 and SP6.

An RNA polymerase according to the invention may possess a proteinsequence homology greater than 50%, and in particular greater than 80%with a wild-type RNA polymerase of the family of DNA-dependent RNApolymerases including the T7 RNA polymerase, T3 RNA polymerase and SP6RNA polymerase.

The abovementioned family of DNA-dependent RNA polymerases is known; seefor example the article by R. Sousa, TIBS 21, 186-190 (1996), and thereferences cited in that article.

Among the polymerases of the invention, there may be mentioned inparticular those which contain at least one mutation in a regioncorresponding to the T7 RNA polymerase sequence containing amino acids625-652, and in particular those which have the composition of awild-type DNA-dependent RNA polymerase, with the exception of the factthat they contain at least one mutation in said region. “Mutation” isunderstood here to mean the replacement, deletion or insertion of anamino acid.

There may be mentioned for example the RNA polymerases containing atleast one mutation at a position corresponding to one of positions 627,628, 631, 632 and 639 of the T7 RNA polymerase amino acid sequence; inparticular said mutation may comprise the replacement of an amino acidresidue, chosen from arginine, lysine, serine and tyrosine, of thewild-type RNA polymerase with another amino acid residue. The amino acidreplaced is for example an arginine or a lysine. The replacement aminoacid may be chosen in particular from alanine, valine, leucine,isoleucine, glycine, threonine or serine. It is understood that theexpression “amino acid” designates here, by a misuse of language, anamino acid residue engaged in a peptide bond.

Reference was made above to the peptide sequence of the T7 RNApolymerase. The numbering of the amino acid residues adopted here isthat described by Dunn, J. J. and Studier, F. W. J. Mol. Biol. 148(4),303-330 (1981), and by Stahl, S. J. and Zinn, K., J. Mol. Biol. 148(4),481-485 (1981).

The invention also relates to:

a gene encoding an RNA polymerase as defined above; such a gene may beobtained for example according to a method similar to that describedbelow in the experimental part;

an expression vector into which such a gene is inserted, said vectorbeing capable of expressing said RNA polymerase in a host cell; thisvector may be obtained in a manner known per se;

a host cell containing such a vector.

The invention also relates to a method of producing an RNA polymerase asdefined above, characterized in that: a) a gene encoding a wild-type RNApolymerase is obtained in a known manner, b) at least one mutation isperformed on said gene, c) the mutated gene obtained is inserted into anexpression vector, d) said vector is expressed in a host cell in orderto obtain a mutated RNA polymerase and e) among the mutated RNApolymerases obtained, those which exhibit at least one of the propertiesof an RNA polymerase as defined above are selected.

A more detailed description of a particular embodiment of the method ofthe invention will be given below in the case of the use of the T7 RNApolymerase as starting material.

A modular gene for T7 DNAdRNAp was prepared, this gene resulting fromthe assembly of different cassettes (see Example 1 and FIG. 1).

The modular gene thus defined is characterized in that it contains 10cassettes bordered by unique restriction sites in the cloning vector.

In particular, these cassettes, bordered by unique restriction sites,are characterized in that each cassette comprises a region of interest,in particular those involved in promoter recognition (region exhibitinghomology with the E. coli a factor; region exhibiting homology with theE. coli σ repressor; region conferring promoter specificity) and thoseinvolved in the catalytic site (motif A; motif B; motif C).

For the definition of motifs A, B and C. see for example R. Sousa, TIBS21, 186-190 (1996).

These cassettes, derived from The T7 DNAdRNAp gene 1, were obtained withthe aid of conventional molecular biology techniques (F. M. Ausubel, R.Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, et al. CurrentProtocols in Molecular Biology (Current Protocols, 1993)), in particularPCR, which made it possible to introduce restriction sites by silentsite-directed mutagenesis, and subcloning into a cloning vector.

The modular gene thus obtained is characterized by the presence ofrestriction sites bordering the cassettes, these restriction sites beingthe Nco I (−2, +4), Bcl I (218, 223), Hind III (539, 544), SacI (776,781), PstI (1587, 1592), BglII (1811, 1816), NdeI (1865, 1870), XhoI(1951, 1956), ClaI (2305, 2310), SalI (2497, 2502), XbaI (2660, 2665)site; position 1, in nucleic acids, corresponds to the adenine of theinitiator ATG codon and position 2652 to the third base of the TAAterminator codon. The NdeI site (2504, 2509) as destroyed. All themutations inducing these restriction sites are silent, except themutation generating the NcoI site which induces the replacement ofasparagine at position +2 with a glycine. Position 1, in amino acids,corresponds to the first methionine, position 883 corresponds to thecarboxy-terminal alanine.

The modular gene, cloned into a cloning vector pGNEX derived from pGEM-1in which the polylinker has been replaced by an adaptor containing theNco I, EcoRI, XbaI restriction sites constitutes the basic support forsubsequent mutageneses. This is made possible by the fact that eachcassette containing a region of interest is bordered by uniquerestriction sites in the cloning vector.

The introduction of nonsilent mutations, with the aid of PCR techniques,into one or more cassettes of the modular gene previously defined, ledto genes encoding polymerases exhibiting an amino acid sequencediffering by at least one amino acid with respect to the T7 expressedfrom the modular gene. Mutant genes were in particular prepared whichencode at least one modified amino acid in the B motif of the wild-typeenzyme, for example with an alanine (A) in place of an arginine (R) atposition 627 and/or an alanine (A) in place of a serine (S) at position628 and/or an alanine (A) in place of a lysine (K) at position 631and/or an alanine (A) in place of an arginine (R) at position 632 and/oran alanine (A) in place of a tyrosine (Y) at position 639.

Mutant genes were also obtained which encode a polymerase whose625VTRSVTKRSVMTLAYGSKEFGFRQQVLD652 (SEQ ID NO: 6) region comprising theB motif has been replaced as a whole or in part by the homologous regionB′ present in some RNA-dependent polymerases, in particular those of thepolymerases of the hepatitis C virus (NCGYRRCRASGVLTTSCGNTLTCYI) (SEQ IDNO:7), and of the yeast integrase 32 (HNTTLGIPQGSWVSPILCNIFLDKL) (SEQ IDNO:8).

The genes described above have been cloned into a vector pMR resultingfrom the ligation of the SspI fragment of pMal-c (Biolabs) containing inparticular the lacI^(q) repressor, and of the SspI fragment of pMH (V.Cheynet, B. Verrier, F. Mallet, Protein expression and purification 4,367 (1993)) containing a minicistron making it possible to achieve ahigh level of expression, as well as a sequence encoding apoly-histidine tail fused with the terminal end of the cloned gene(Example 1, FIG. 2). The expression of the recombinant proteins T7 RNApinto the bacterial strain BL21 represents up to 30% of the totalproteins of the bacterium. The proteins solubilized in a deoxycholatebuffer containing a high salt concentration are deposited on a TALONcolumn (Clontech) allowing specific purification by chelation with theion of proteins having a poly-histidine tail. 130 to 2200 μg ofpolymerases are thus obtained for 20 ml of culture, with a puritygreater than 95% as indicated (i) by a Coomassie blue staining (Example1, FIG. 3), (ii) by a Western-blot analysis with a guinea-pig polyclonalantibody anti-T7 RNAP (bioMérieux) and a mouse monoclonal antibody(Quiagen) anti-MRGSHHHHHH (SEQ ID NO: 9), (iii) by the absence ofendonuclease, single-stranded and double-stranded exonuclease orribonuclease activity, as determined essentially according to the methodof He et al (B. A. He, M. Rong, D. L. Lyakhov, H. Gartenstein, G. Diaz,et al, Protein Expr Purif 9, 142 (1997)). This result reflects theperformances of the host-pMR-BL21 vector pair and of the method ofpurification with respect to the MRGSHHHHHSVLE (SEQ ID NO: 10) tail.

The subject of the invention is also the use of an RNA polymerase asdefined above, in a method of transcription of a polynucleotide segmentof interest having any sequence, said segment, of the RNA type, beingcontained in a polynucleotide template, so as to synthesize, in thepresence of said template, a product of transcription containing an RNAsequence complementary to the sequence of said polynucleotide segment ofinterest.

According to a particular embodiment, said use is characterized in thatsaid polynucleotide template comprises, upstream of said polynucleotidesegment of interest, a promoter recognized by said RNA polymerase, andin that said product of transcription is an RNA complementary to asequence of the template starting at a site of initiation oftranscription for said promoter.

The RNA polymerases of the invention may be used in particular to carryout (i) an amplification of an RNA target isothermally, (ii) a directsequencing of, RNA and (iii) the synthesis of RNA of special interest(for example probes, ribozymes and the like). In addition, RNApolymerases of the invention are capable of incorporating modified basesinto the newly-synthesized strand, which facilitates in particular thequantification or the use of said strand.

The invention relates in particular to the use of these recombinantenzymes thus expressed and purified in a method of synthesizing RNA froman RNA template, under the control of a promoter.

The enzymes thus purified were evaluated, in a promoter-dependentcontext, on different templates (Example 2, FIG. 4 on the one hand, andExample 3, FIG. 6, on the other hand) in particular a templatecontaining a single-stranded RNA. It has been shown that a mutatedpolymerase obtained according to the invention was capable of generatinga specific transcript of the correct size in particular on asingle-stranded RNA template. In Example 2, it is indicated that thewild-type enzyme identically produced does not appear to be able tocarry out this phenomenon. However, Example 3 shows that the wild-typeenzyme in fact possesses the property of transcribing an RNA template.These apparently divergent results are explained by the fact that theexperimental conditions in these two examples are different. Indeed, inExample 2, the presence of the transcript is identified by the techniqueof incorporating a UTP, labeled with radioactive phosphorus, whereas inExample 3 the technique used is Northern blotting for the group 2templates, and in the latter case, the detection of the transcript bythe Northern blotting technique is 40 times more sensitive than thedetection by incorporation of radioactive phosphorus. Example 2 belowtherefore shows that a mutated polymerase obtained according to theinvention is capable of generating a specific transcript of the correctsize on a single-stranded RNA template, and Example 3 shows that in factthe corresponding wild-type polymerase is capable of generating aspecific transcript of the correct size on RNA templates independentlyof their sequence.

Furthermore, such a mutated polymerase is incapable, unlike thewild-type polymerase, of generating a transcript of the correct size ona single-stranded or double-stranded DNA template. If the Mg²⁺ ionpresent in the reaction medium is replaced by the Mn²⁺ ion, the mutatedenzyme, on a single-stranded RNA template, does not generate under theseconditions a specific transcript of the correct size, but isnevertheless capable of generating large quantities of abortiveproducts. Such a mutated polymerase is in addition capable of displacingan RNA/RNA hybrid.

In the appended drawings:

FIG. 1 illustrates the amplification strategy, described in detail inExample 1 below, for the construction of a modular gene for the T7 RNApolymerase,

FIG. 2 illustrates the structure of the expression vector pMR containingthis modular gene,

FIG. 3 represents electrophoretic profiles of the proteins expressedwith the aid of this vector, as described in detail in Example 1 below,

FIG. 4 schematically represents the template systems used in thetranscription trials of Example 2 below,

FIG. 5 represents the electrophoretic profiles of the products oftranscription obtained in Example 2 below, and

FIG. 6 schematically represents the template systems used in thetranscription trials of Example 3 below.

The following examples illustrate the invention.

EXAMPLES Example 1

Construction of the Gene for T7 RNA Polymerase in Modular Form;Expression and Purification of the T7 RNA Polymerase

The T7 RNA polymerase gene was constructed in modular form, taking intoaccount the regions of homology with the other polymerases as well asthe functions associated with certain domains of T7 RNA polymerase. Itwas divided into 10 regions or cassettes (FIG. 1-A), each region beingdelimited by unique restriction sites in the cloning vector. Sixcassettes are characteristic in that they contain respectively a domainhaving similarity with part of the E. coli sigma factor, a domain havinghomology with the E. coli lambda repressor, a domain involved in thephage polymerase promoter specificity, the A and C motifs conserved inthe template-dependent polymerases and the B motif conserved in theDNA-dependent polymerases. Each region is amplified by PCR (F. M.Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, et al,Current Protocols in Molecular Biology (Current Protocols, 1993)) (FIG.1a-B1) from the wild-type gene (gene 1) using primers defined asfollows: (i) these primers contain silent mutations allowing theintroduction of restriction sites delimiting the cassettes; (ii) theprimer situated at position 3′ of one region and the primer situated atposition 5′ of the contiguous region partially overlap. The contiguousamplification products are mixed and reamplified using external primers(FIG. 1a-B1). The 5 fragments generated are cloned into the vector PCRII(Invitrogen) (FIG. 1-B2). From the 5 cloned fragments, the T7 RNApolymerase gene was reconstructed from the 5′ end to the 3′ end bysuccessive subclonings into the cloning vector PGNEX (FIG. 1-B3). Thisvector is derived from the plasmid pGEM-1 (Promega) in which thepolylinker is replaced by an adaptor containing the NcoI-EcoRI-XbaIrestriction sites. The modular gene for T7 RNA polymerase (T7 RNA pol)thus obtained contains 17 unique cloning sites in pGNEX of which 11newly-introduced sites (FIG, 1-C). This gene was then subcloned into theexpression vector pMR (FIG. 2). This vector, derived from the vector pMH(V. Cheynet, B. Verrier, F. Mallet, Protein expression and purification4, 367 (1993)), contains the strong tac promoter regulated by IPTG(isopropyl-beta-D-thiogalactopyranoside), a short minicistron surroundedby two ribosome-binding sites constituting an efficient context forinitiation of translation and a polyhistidine tail fused with theN-terminal end of the expressed protein allowing its purification bymetal ion-affinity chromatography. The gene encoding the lacI^(q)repressor allowing a better control of expression is also present inpMR. This expression vector is transformed according to conventionalmethods (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G.Seidman, et al, Current Protocols in Molecular Biology (CurrentProtocols, (1993)) in the E. coli BL21 bacterial strain. A preculture isused to inoculate 30 ml of culture medium. The expression of theproteins is induced by IPTG for 4 h at 37° C. as soon as the opticaldensity at 600 nm of the culture reaches 0.6. The T7 RNA pol thusexpressed represents 30% of the total bacterial proteins (FIG. 3). It isthen extracted from the bacteria by lysis (sonication) in the presenceof a detergent. From the soluble extracted fraction, the T7 RNA pol ispurified by Co²⁺-ion affinity chromatography and eluted with animidazole gradient. It is analyzed on an SDS-PAGE gel and visualized byCoomassie blue staining (FIG. 3). The absence of degradation products isverified by Western blotting with a polyclonal antibody anti-T7 RNA polproduced in guinea-pigs and a monoclonal antibody anti-MRGSH₆ which iscommercially available (Quiagen). The absence of spurious endonuclease,exonuclease and RNase activities is verified according to the protocoldescribed by He et al, Protein Expr Purif 9, 142 (1997)). From 20 ml ofculture 700 to 800 μg of enzymes, having a purity greater than 95%, areobtained in a reproducible manner. The purification yield is 75%.

The point mutations are created by sequential PCR (F. M. Ausubel, R.Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, et al, CurrentProtocols in Molecular Biology (Current Protocols, (1993)) (or doublePCR), in the same manner in which the restriction sites were introduced:the internal primers contain the mutation to be introduced; the externalprimers flank the region to be mutated and contain the restriction sitesdelimiting this region in the modular gene. This cassette thus mutatedis then cloned into the modular gene. Synthetic oligonucleotides mayalso replace a complete cassette.

Legend to the Figures Mentioned in Example 1

FIG. 1: illustrates the amplification strategy for the construction ofthe modular gene for the T7 RNA polymerase. (A): Division of the T7 RNApolymerase gene (vertical arrows) with respect to the regions ofinterest. The regions marked from 1 to 6 are: 1, homology with part ofthe E. coli sigma factor; 2, homology with the E. coli lambda repressor;3, motif A; 4, motif B; 5, phage polymerase promoter specificity; 6,motif C. (B): (1) Position of the primers containing the silentmutations for the creation of the restriction sites (-□-); Nc, NcoI; Bc,BclI; H, HindIII; Sc, SacI; P, PstI; Bg, BglII; Nd, NdeI; Xo, XhoI; C,ClaI; Sl, SalI; Xb, XbaI. Extension by overlapping PCR leads to 5fragments containing 3 restriction sites (F1 to F5). Sub-cloning means:subcloning. (2) Each fragment thus generated (F1 to F5) is cloned intothe vector PCRII. E, EcoRI. (3) Reconstruction of the modular gene fromthe 5′ end to the 3′ end, using at each step the restriction site commonto 2 fragments and the EcoRI restriction site of the vector PCRII and ofthe cloning vector pGNEX; the XbaI restriction site common to the lastfragment and to the vector pGNEX is used for the last subcloning. (C)structure of the modular gene for T7 RNA polymerase and position of theunique restriction sites in the vector pGNEX which already exist (at thebottom) and created by mutagenesis (at the top).

FIG. 2: illustrates the structure of the expression vector pMRcontaining the modular gene for T7 RNA polymerase. Ptac: tac promoter(black box); RBS1-MC-RBS2, minicistron surrounded by 2 ribosome-bindingsites (white arrow); (His)6T7RNAPcas, modular gene for T7 RNA polymerase(gray arrow); rrnB T1 T2, strong transcription terminators (box withdotted lines); bla, gene for resistance to ampicillin (black arrow);pMB1 ori/M132 ori-, replication origins (thin white box); lacI^(q), geneencoding the lacI^(q) repressor (hatched arrow); some restriction sitesare indicated, including the new sites introduced by mutagenesis(underlined).

FIG. 3: shows the electrophoretic profiles obtained allowing analysis ofthe expression and of the purification of the T7 RNA polymerase. Thebacterial lysates are prepared from 1 ml collected from 30 ml of culturein order to verify the expression of T7 RNA polymerase in the presence(+) or absence (−) of IPTG. The T7 RNA polymerase is extracted from 20ml of the same culture. The soluble fraction is loaded onto thepurification column (lane L) and the T7 RNA polymerase is eluted (laneE) with an imidazole gradient. M, molecular weight marker. The proteinsare visualized on a 10% SDS-PAGE gel by Coomassie blue staining. Thearrow indicates the position of the T7 RNA polymerase.

Example 2

Transcription of RNA and DNA Templates by the Mutated T7 RNA PolymeraseR627A Under the Control of a Promoter

The template systems used in this example are schematically presented inFIG. 4. The single-stranded DNA transcription system comprises atemplate strand (a) of 50 bases having the sequence3′ATTATGCTGAGTGATATCCCAACCGGCGTCACAAGTGAGTACCAATACCG5′ (SEQ ID NO: 2)and hybridized from positions −17 to +1 with the non-template promoterstrand (b) having the sequence 5′TAATACGACTCACTAG 3′ (SEQ ID NO: 11)(blocked at its 3′ end). The double-stranded DNA transcription systemcomprises the template strand (a) hybridized with the non-templatestrand (c) having the sequence5′TAATACGACTCACTATAGGGTTGGCCGCAGTGTTCACTCATGTTATGGC3′ (SEQ ID NO: 12).

The single-stranded RNA transcription system consists of a DNA hybridstrand from positions −17 to −1 and RNA from positions +1 to +33 havingthe sequence 3′ATTATGCTGAGTGATATCCCAACCGGCGUCACAAGUGAGUACCAAUACCG5′ (SEQID NO: 13), hybridized with the non-template promoter strand (b). Theexpected complete transcripts on the three systems are of 33 bases.

The reactions are performed in 20 μl of a buffer derived from thatdescribed by J. F. Milligan, D. R. Groebe, G. W. Witherell, O. C.Uhlenbeck, Nucleic Acids Res. 15, 8783 (1987), namely Tris-HCl 40 mM, pH8.1, spermidine 1 mM, PEG 8% (g/V), TRITON X-100 (Octoxynol-9) 0.01%(V/V), BSA 5 μg/100 μl, 1 μl (40 u) of porcine RNAguard (PharmaciaBiotech), UTP 12.5 μM, a 32P UTP 0.5 μCi (Amersham, 10 mCi/ml 400Ci/mmol) 0.4 mM of the three ribonucleoside triphosphates A, G, C,Mg(OAc)₂ 6 mM. The template concentration is set at 10¹¹ copies of eachstrand in 20 μl of reaction. The wild-type T7 RNA polymerase is used at0.5 μM (100 ng/20 μl), the mutated T7 RNA polymerase R627A at 3.65 μM(730 ng/20 μl). Before adding the enzymes, the reactions are denaturedfor 5 minutes at 65° C. in a heating block and then gradually brought to37° C. The reactions are initiated by the addition of the polymerases,incubated for 1 hour at 37° C. and then stopped by the addition of anequal volume of 2× blue formamide (formamide 90%, EDTA 25 mM, xylenecyanol 0.02%, bromophenol blue 0.02%) and denatured for 5 minutes at 95°C. 20 μl of each reaction are deposited on a denaturing gel (20%acrylamide, urea 7 M, 1×TBE), and then after migration, the gel isautoradiographed at −70° C. on a Biomax MR film (Kodak). The results(electrophoretic profiles) are presented in FIG. 5, and in particularthe transcription results obtained with the mutated T7 RNA polymeraseR627A (wells 1-3) and the wild-type T7 RNA polymerase (wells 4-6), onthe single-stranded RNA templates (wells 1 and 4), double-stranded DNA(wells 2 and 5), and single-stranded DNA (wells 3 and 6). Thetranscription on single-stranded RNA, detected by detection of acomplete transcript of 33 bases, is possible using the mutated T7 RNApolymerase R627A (well 1) and not the wild-type enzyme (well 4) whichproduces on the other hand many abortive transcripts; see neverthelessthe different results obtained in Example 3 below. The mutated T7 RNApolymerase R627A exhibits a residual transcription activity ondouble-stranded DNA (well 2), characterized by the presence of apredominant transcript which is smaller in size than the expectedtranscript, and the presence of a small quantity of abortive products.On single-stranded DNA (well 3), this transcript of abnormal sizedisappears, whereas the quantity of abortive products increases. Bycontrast, the wild-type enzyme allows the production of specifictranscripts in the presence of DNA templates (wells 5 and 6), thisenzyme exhibiting, moreover, a better transcription activity on thedouble-stranded DNA template (well 5) than on the single-stranded DNAtemplate (well 6); for these two templates, the wild-type enzyme inducesthe synthesis of numerous abortive transcripts. These results show thatthe replacement of the arginine 627 by an alanine confers on the mutantenzyme the possibility of synthesizing RNA from an RNA template andinduces the loss of capacity to synthesize RNA from a DNA template.

Example 3

The T7 RNA polymerase obtained as in Example 1 is purified by affinitychromatography according to the method described by Arnaud N. et al.,Gene, 199, 149-156 (1997).

The purified T7 RNA polymerase has a specific activity of about 200U/μg.

The sequences of the templates used for the transcription are describedin Table 1 below.

In the trials described below, the templates of sequence 1, 2 and 3 ofTable 1 are called group 1, group 2 and group 3 templates, respectively.

The sequence of the probes used is represented in Table 1 (sequencesNos. 4 and 5).

Sequence No. 4 recognizes the 3′ end of the products of transcriptionobtained with the templates of groups 1 and 3, and sequence No. 5recognizes the 3′ end of the products of transcription of the group 2template.

The probes are labeled with γ³²P ATP using T4 polynucleotide kinase orwith a α³²P ddATP using a deoxynucleotide terminal transferase.

Transcriptional Trials

The reactions are carried out in a volume of 20 μL containing 40 m/MTris-HCl, 1 mM spermidine 50 μg/ml of bovine serum albumin, 0.01% (v/v)Triton X100, 80 mg/ml PEG 8000, 1 μl RNAguard (Pharmacia), 6 mMmagnesium acetate, 10¹¹ copies of template and non-template strands, thenucleoside triphosphates necessary for the transcription and thenucleoside triphosphates labeled as indicated. When the labelednucleoside triphosphate is α³²P ATP, concentrations of 0.4 mM UTP, CTPand GTP, 12.5 μM ATP and 0.5 μCi α³²P ATP (New England Nuclear-Dupont,800 Ci/mmol) are used. When the labeled nucleoside triphosphate is α³²PUTP, concentrations of 0.4 mM ATP, CTP and GTP, 12.5 μM of ATP and 0.5μCi α³²P UTP (Amersham, 400 Ci/mmol) are used. When the labelednucleoside triphosphate is γ³²P GTP, concentrations of 0.4 mM ATP, UTP,CTP and GTP, and 20 μCi γ³²P GTP (Amersham, 5000 Ci/mmol) are used.

The samples are heated for 5 minutes at 70° C. and then slowly cooled to37° C. in order to allow the hybridization of the template andnon-template strands. 260 ng of T7 RNA polymerase are then added and themixture is incubated for 1 hour at 37° C. The reactions are stopped byadding an equal volume of 2×buffer (90% formamide, 25 mM EDTA, 0.02%xylene cyanol, 0.02% bromophenol blue). The products of transcriptionare analyzed by electrophoresis, after heating for 5 minutes at 95° C.,on a 20% denaturing polyacrylamide gel, and examined by autoradiography.

For the Northern blot analysis, the reactions are carried out under thesame conditions, but without the labeled nucleotides. After migration ona 20% denaturing polyacrylamide gel, the samples are transferred ontonylon membrane (Appligene) and the products of transcription aredetected by the appropriate labeled probe and are examined byautoradiography.

Definition of Short Synthetic Templates

Three types of short synthetic templates, containing a double-strandedDNA promoter, were defined in order to verify if the T7 RNA polymeraseis capable of using an RNA template in a transcription reaction.

The first type of templates (RNA+18) was defined so as to study thecapacity of the T7 RNA polymerase to transcribe an RNA template duringthe so-called processive phase. This first type of template comprises adouble-stranded promoter followed downstream by a chimeric DNA-RNAsequence whose transition is situated 18 bases downstream of the site ofinitiation of transcription.

The second type of templates (RNA+1) was defined so as to study thecapacity. of the T7 RNA polymerase to transcribe an RNA template duringthe start of transcription phase. This second type of templatescomprises a double-stranded promoter followed by an RNA sequence.

The third type of templates (DNA), which serves as a comparison,comprises a double-stranded promoter followed downstream by a DNAsequence.

To study the influence of the non-template strand, the DNA, RNA+18 andRNA+1 templates may be either single-stranded (m), or double-strandedheteroduplexes (bhe), or double-stranded homoduplexes (bho). Thedouble-stranded homoduplex template RNA+18 forms an RNA-RNA duplexstarting from position +18. Likewise, the double-stranded homoduplextemplate RNA+1 forms an RNA-RNA duplex starting from the transcriptionalstart site.

It should be noted that in Table 1, the different nucleotides have beenrepresented with the letters A, T, C, G. When the template, or part ofthe template, is DNA, these letters represent deoxyribonucleotides. Whenthe template, or part of the template, is in the form of RNA, theseletters represent ribonucleotides and it should be understood that thesymbol T is then used in place of the symbol U.

The different transcription systems which have just been mentioned areschematically represented in the appended FIG. 6 in which, for eachdouble-stranded system, the top strand is the non-template strand andthe bottom strand is the template strand. The DNA double-strandedpromoter region is represented by a thick line. The DNA strands arerepresented by a thin line, the RNA strands are represented by adiscontinuous line. The expression +1 corresponds to the transcriptionalstart site. The expression +18 corresponds to the eighteenth basedownstream of the transcriptional start site. In the representation ofthese various transcription systems, the letter m means single-stranded;the letter b means double-stranded; bhe means double-strandedheteroduplex; and bho means double-stranded homoduplex.

The transcription systems of FIG. 6 were used with each of thetemplates, or, depending on the cases, with some of the templates ofTable 1. The group 1 templates, using sequence No. 1, correspond to adouble-stranded DNA promoter sequence followed by a consensus region ofinitiation +1 to +6 (Dunn and Studier, J. Mol. Biol., 166, 477-535,1983), itself followed by a sequence of twenty-six bases downstream.

The group 2 templates correspond to a double-stranded DNA promotersequence followed by a nonfavorable sequence, since it can allow theearly termination of transcription (Martin et al., Biochemistry, 27,3966-3974, 1988).

The group 3 templates correspond to the same nonfavorable initiationsequence as group 2, followed by the same downstream sequence as group1.

Results

The results are summarized in the accompanying Table 2.

It is observed that the DNA-RNA transition on the RNA+18 template isefficiently passed via the elongation complex, as shown by the absenceof an increase in early terminations around position +18, compared withthe DNA control.

The T7 RNA polymerase is capable of initiation of transcription ondifferent RNA templates and is capable of fully transcribing thesetemplates. The detection of transcripts having the expected size, forall the groups of templates, shows that the use of the RNA template isnot dependent on the sequence, although the overall efficiency dependson the base composition, as is also observed on the control DNAtemplates.

The different results mentioned in Example 2 above result from the factthat the detection by the Northern blotting technique is in the presentcase 40 times more sensitive than the detection technique used inExample 2, as was indicated above in the description.

The T7 RNA polymerase is capable of transcribing an RNA-RNA duplex. Noincrease in the number of abortive products of transcription is observedwith the RNA+1 homoduplex template.

The transcription of the homo- or heteroduplex double-stranded templatesis similar overall. In the case of the group 2 RNA+18 templates, moretranscripts of the expected size are obtained with the homoduplex systemthan with the heteroduplex system.

The presence of the non-template strand influences the efficiency oftranscription. Indeed, the yield of transcription is increased on thesingle-stranded templates, as shown, on the one hand, by the increase inthe number of transcripts of the correct size and, on the other hand, bythe fact that an initiation event is more frequently associated with thesynthesis of a complete transcript. However, with the group 2 templates,the single-stranded system does not give better results.

Thus, the T7 RNA polymerase possesses transcriptional activity on RNAtemplate, in the presence of a DNA double-stranded promoter. The yieldof transcription observed is only 10 to 100 times lower than on DNAtemplate.

TABLE 1 SEQUENCE No. 1:3′ ATTATGCTGAGTGATATCCCTCTAGATCGTCGTATTGCGAGGTTCATGT 5′                     +1              +18 SEQUENCE No. 2:3′ ATTATGCTGAGTGATATCCCAACCGGCGTCACAAGTGAGTACCAATACCG 5′                     +1              +18 SEQUENCE No. 3:3′ ATTATGCTGAGTGATATCCCAACAGATCGTCGTATTGCGAGGTTCATGT 5′                     +1              +18 SEQUENCE No. 4:3′ ATTGCGAGGTTCATGT 5′ SEQUENCE No. 5: 3′ GTGAGTACCAATACCG 5′

TABLE 2 Group (1) Group (2) Group (3) Template TS^(a) EI^(b) Eff^(c)TS^(a) EI^(b) Eff^(c) TS^(a) EI^(b) Eff^(c) DNA m 240 242 1/10 192 5931/31 149 633 1/43 DNA bho 80 152 1/18 102 191 1/19  67 893 1/133 RNA +18 m 800 650 1/8 326 1488  1/45 253 1246  1/49 RNA + 18 208 309 1/14  99647 1/65  61 642 1/106 bhe RNA + 18 176 272 1/16 358 1093  1/31 nr^(e)nr nr bho RNA + 1 m 13 141 1/108 NA^(f) NA NA NA^(d) NA NA RNA + 1 bhe3.2  61 1/227 NA^(d) NA NA NA^(d) NA NA RNA + 1 bho 6.4 110 1/157 NA^(d)NA NA nr nr nr Comparison of transcription by the T7 polymerase ondifferent templates, by incorporation of γ³²P-GTP ^(a)Ts: specifictranscript in picomoles per 10 picomoles of template ^(b)EI: number ofinitiation events for one copy of template ^(c)Eff: the efficiencycorresponds to the number of initiation events leading to the synthesisof a complete transcript NA: not accessible (nonquantifiable) d: notaccessible; however the complete transcript is detected in Northernblotting nr: not done

13 1 49 DNA Artificial Sequence Description of Artificial SequenceTranscription Template 1 tgtacttgga gcgttatgct gctagatctc cctatagtgagtcgtatta 49 2 50 DNA Artificial Sequence Description of ArtificialSequence Transcription Template 2 gccataacca tgagtgaaca ctgcggccaaccctatagtg agtcgtatta 50 3 49 DNA Artificial Sequence Description ofArtificial Sequence Transcription Template 3 tgtacttgga gcgttatgctgctagacaac cctatagtga gtcgtatta 49 4 16 DNA Artificial SequenceDescription of Artificial Sequence Probe 4 tgtacttgga gcgtta 16 5 16 DNAArtificial Sequence Description of Artificial Sequence Probe 5gccataacca tgagtg 16 6 28 PRT Escherichia coli 6 Val Thr Arg Ser Val ThrLys Arg Ser Val Met Thr Leu Ala Tyr Gly 1 5 10 15 Ser Lys Glu Phe GlyPhe Arg Gln Gln Val Leu Asp 20 25 7 25 PRT Hepatitis C Virus 7 Asn CysGly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser 1 5 10 15 CysGly Asn Thr Leu Thr Cys Tyr Ile 20 25 8 25 PRT Yeast Integrase 8 His AsnThr Thr Leu Gly Ile Pro Gln Gly Ser Val Val Ser Pro Ile 1 5 10 15 LeuCys Asn Ile Phe Leu Asp Lys Leu 20 25 9 10 PRT Artificial SequenceDescription of Artificial Sequence Antigen of a Mouse MonoclonalAntibody 9 Met Arg Gly Ser His His His His His His 1 5 10 1 10 13 PRTUnknown Organism Description of Unknown Organism Poly-histidine Tail 10Met Arg Gly Ser His His His His His Ser Val Leu Glu 1 5 10 11 18 DNAArtificial Sequence Description of Artificial Sequence Non-templatePromoter Strand 11 taatacgact cactatag 18 12 50 DNA Artificial SequenceDescription of Artificial Sequence Non-template Strand 12 taatacgactcactataggg ttggccgcag tgttcactca tggttatggc 50 13 50 DNA ArtificialSequence Description of Artificial Sequence Transcription Template 13gccauaacca ugagugaaca cugcggccaa ccctatagtg agtcgtatta 50

What is claimed is:
 1. A method of amplifying an RNA target sequence inan RNA sample comprising said target sequence, said method comprising:(A) bringing said sample into contact: with a reagent capable ofhybridizing with RNA comprising said target sequence, in the absence ofdeoxyribonucleoside triphosphates, and with an enzymatic systemcomprising an RNA polymerase, under conditions allowing thehybridization of said reagent with said RNA comprising said targetsequence and under conditions allowing the functioning of said RNApolymerase; wherein said reagent contains: (i) a first nucleotide strandcomprising: a) a first nucleotide segment capable of playing the role ofsense strand of a promoter for said RNA polymerase and b) downstream ofsaid first segment, a second nucleotide segment comprising a sequencecapable of hybridizing with a region of said RNA, and (ii) in thehybridized state on the first strand, a second nucleotide strandcomprising a third nucleotide segment capable of hybridizing with saidfirst segment so as to form with it a functional double-strandedpromoter; and wherein said RNA polymerase (1) is a T7-like phage RNApolymerase and (2) is capable of transcribing an RNA template, in thepresence of said reagent hybridized with said template, in the absenceof associated protein factor and in the absence of a ligase activity;and (B) amplifying said target sequence by transcription under controlof the double-stranded promotor.
 2. A method according to claim 1,wherein said third segment is flanked, at its upstream end, by a fourthnucleotide segment which is shorter than said second segment of thefirst strand.
 3. A method according to claim 2, wherein said fourthsegment is capable of hybridizing with a portion opposite said secondsegment.
 4. A method according to claim 2, wherein said fourth segmentof said second strand is chosen from those whose sequence facilitatesthe initiation of transcription for said RNA polymerase.
 5. A methodaccording to claim 2, wherein said second segment of said first strandcontains a number of nucleotides at least equal to the sum of the numberof nucleotides of said fourth segment, if it is present, and of thenumber of nucleotides of said sequence of the second segment which iscapable of hybridizing with said region of said RNA.
 6. A methodaccording to claim 2, wherein said fourth segment consists of DNA. 7.The method according to claim 2, wherein said fourth segment contains 1to 18 nucleotides.
 8. The method according to claim 7, wherein saidfourth segment contains 1 to 12 nucleotides.
 9. A method according toclaim 1, wherein said first and third segments consist of DNA.
 10. Amethod according to claim 1, wherein said T7-like phage RNA polymeraseis selected from the group consisting of T7 RNA polymerase, T3 RNApolymerase and SP6 RNA polymerase.
 11. A method according to claim 1,wherein said T7-like phage RNA polymerase is derived by mutation from anRNA polymerase selected from the group consisting of T7, T3 and SP6 RNApolymerases.
 12. A method according to claim 11, wherein said RNApolymerase contains at least one mutation in the region corresponding tothe T7 RNA polymerase sequence containing amino acids 625 to 652 (SEQ IDNO: 6).
 13. A method according to claim 12, wherein said RNA polymeraseis capable of transcribing a polynucleotide target sequence with ahigher yield when said target sequence consists of RNA than when itconsists of DNA.
 14. A method according to claim 1, wherein said enzymesystem contains only RNA polymerase activity.
 15. The method ofamplifying an RNA target sequence according to claim 1, whereinpromoters of the T7-like phage RNA polymerase have a consensus sequencefrom position −17 to position −1, relative to position +1 being the siteof initiation of transcription.
 16. The method according to claim 1,wherein said first segment contains at least 9 nucleotides.
 17. Themethod according to claim 1, wherein said first segment contains atleast 6 nucleotides.